Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.
Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.
复制标题
酵母转录因子 IID 中的两个不同结构域以及 TATA 盒诱导的构象变化的证据。
DOI：
10.1128/mcb.11.1.63-74.1991
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发表时间：
1991
影响因子：
5.3
通讯作者：
Berk,AJ
中科院分区：
文献类型：
--
作者：
Lieberman,PM;Schmidt,MC;Kao,CC;Berk,AJ
来自酿酒酵母 (YIID) 的转录因子 IID 与大多数 RNA 聚合酶 II 启动子中存在的 TATA 盒元件结合。在这项工作中，部分蛋白水解被用作YIID结构的生化探针。 YIID 由蛋白酶敏感的氨基末端和高度稳定、耐蛋白酶的羧基末端核心组成。通过氨基末端肽测序，将主要胰凝乳蛋白酶和胰蛋白酶衍生片段的切割位点分别定位到氨基酸残基40至41和48至49。氨基末端的去除导致 YIID 在凝胶电泳迁移率变动测定中与 DNA 形成稳定复合物的能力显着增加，并且通过 DNase I 足迹分析测定，DNA 结合亲和力增加了两到四倍。羧基末端 190 个氨基酸核心能够进行体外转录，并且活性与天然 YIID 相似。含有 TATA 元件的 DNA 在氨基末端结构域中诱导超敏感位点，并稳定核心结构域以进一步进行蛋白水解攻击。天然 YIID 在 0°C 时不与 TATA 盒结合，而羧基末端 DNA 结合域却可以。这些结果表明，YIID 在与 TATA 盒结合后发生构象变化。 Southern印迹表明，羧基末端结构域高度保守，而氨基末端结构域在进化过程中迅速分化，即使在密切相关的芽殖酵母之间也是如此。
Transcription factor IID fromSaccharomyces cerevisiae(YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0°C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.